The present invention relates to the field of lipid vesicle technology. More particularly, the present invention discloses lipid vesicles made of surfactants and steroids, possibily with other additives, and a method for their manufacture.
Liposomes, or lipid vesicles made using phospholipids, have been known since at least 1965. There are three general types of lipid vesicles: multilamellar vesicles (MLV), onion-like structures having a series of substantially spherical shells formed of lipid bilayers interspersed with aqueous layers; large (greater than 0.45.mu. diameter, preferably greater than 1.mu. diameter) unilamellar vesicles (LUV) which have a lipid bilayer surrounding a large, unstructured aqueous phase; and small unilamellar vesicles (SUV) which are similar in structure to the LUV's except their diameters are less than 0.2.mu.. Because of the relatively large amount of lipid in the lipid bilayers of the MLV's, MLV's are considered best for encapsulation or transportation of lipophilic materials whereas the LUV's, because of their large aqueous/lipid volume ratio, are considered best for encapsulation of hydrophilic molecules, particularly macromolecules. SUV's have the advantage of small size, which allows relatively easy access to the cells of tissue, but their small volume limits delivery of hydrophilic or aqueous materials to trace amounts. SUV's are more useful in the transportation of lipophilic materials.
As noted, all of the early lipid vesicle studies used phospholipids as the lipid source for the bilayers. The reason for this choice was that phospholipids are the principal structural components of natural membranes. However, there are many problems using phospholipids for liposome structures. First, isolated phospholipids are subject to degradation by a large variety of enzymes. Second, the most easily available phospholipids are those from natural sources, e.g., egg yolk lecithin, which contain polyunsaturated acyl chains that are subject to autocatalyzed peroxidation. When peroxidation occurs, the liposome structure breaks down, causing premature release of encapsulated materials and the formation of toxic peroxidation byproducts. This problem can be avoided by hydrogenation but hydrogenation is an expensive process, thereby raising the cost of the starting materials. Third, cost is a problem associated with the use of phospholipids on a large scale. A kilogram of egg yolk lecithin pure enough for liposome production presently costs in excess of $40,000. This is much too high a cost for a starting material for most applications.
Because of the high cost and additional problems in using phospholipids, a number of groups attempted to use synthetic amphiphiles in making lipid vesicles. For example, Vanlerberghe and others working for L'Oreal have used a series of synthetic polymers, primarily polyglycerol derivatives, as alternatives to the phospholipids. Similarly, Kelly and a group at Sandoz, Inc. have tried aliphatic lipids.
Recently, there has been some indication, particularly from the L'Oreal group, that surfactants might be used to form the lipid bilayer in liposome-like multilamellar lipid vesicles. Both surfactants and phospholipids are amphiphiles, having at least one lipophilic acyl or alkyl group attached to a hydrophilic head group. Head groups in surfactants, which are attached to one or more lipophilic chains by ester or ether linkages, include hydrophilic molecules such as polyoxyethylene, sorbitan, and polyglycerol derivatives. Commercially available surfactants include the BRIJ family of polyoxyethylene acyl ethers, the SPAN sorbitan alkyl esters, and the TWEEN polyoxyethylene sorbitan fatty acid esters, all available from ICI Americas, Inc. of Wilmington, Del.
Substantially all of the surfactants tried for lipid vesicle formation have relatively short (eighteen or under) carbon chains. This is because as the carbon chains grow too long, the lipophilic or hydrophobic regions are bulky so they do not easily form close packed lipid bilayers.
The experiments reported in the literature using synthetic surfactants rather than phospholipids to make multilamellar lipid vesicles have not shown any improvement in the ability to encapsulate either small or large hydrophilic molecules nor is there any increased stability of the lipid vesicles. In particular, there is no indication that lipid vesicles manufactured with these synthetic materials are particularly useful to achieve the hydrophilic and macromolecule delivery objects sought.
No matter what starting material is used to form the MLV's, substantially all of the methods of vesicle production reported in the literature use either the original Bangham method, as described in Bangham et al., J. Mol. Biol., 13: 238-252 (1965), or a minor variation. This basic approach starts by dissolving the lipids, together with any other lipophilic substances including any cholesterol used, in an organic solvent. The organic solvent is removed by evaporation, either using heat or by passing a stream of an inert gas (e.g., nitrogen) over the dissolved lipid to remove the solvent. The residue is then slowly hydrated with an aqueous phase, generally containing electrolytes and any hydrophilic biologically active materials, to form large multilamellar lipid membrane structures. In some variations, different types of particulate matter or physical structures have been used during the evaporation step to change the properties of the lipophilic phase and assist in the formation of the lipid residue. The basis for the use of these particulates is that by changing the physical structure of the lipid residue, better vesicles may form upon hydration. Two recent review publications, Szoka and Papahdjopoulos, Ann. Rev. Biophys. Bioeng. 9: 467-508 (1980), and Dousset and Douste-Blazy, in Les Liposomes, Puisieux and Delattre, Editors, Tecniques et Documentation Lavoisier, Paris, pp. 41-73 (1985), summarizes many of the methods which have been used to make MLV's.
Onde the MLV's are made, it is helpful to determine the effectiveness of the process. Two common measurements for effectiveness of encapsulation are encapsulated mass and captured volume. Encapsulated mass is simply the mass of substance encapsulated per unit mass of lipid, normally given in g material encapsulated/g lipid, or merely as a percentage. The captured volume is a measure of the water content trapped within the vesicles. The captured volume is defined as the amount of the aqueous fraction inside the vesicle divided by the total amount of lipid in the vesicle, normally given in ml/g lipid.
Multilamellar lipid vesicles made using the classic materials and methods have low encapsulated mass for hydrophilic materials, normally in the order of 5-15%. In addition, the captured volume is normally in the order of 2-4 ml/g lipid. However, the encapsulated mass for lipophilic materials is much better. Therefore, multilamellar lipid vesicles made using these standard procedures are good for encapsulating lipophilic (hydrophobic) materials, but are not as good for hydrophilic encapsulation.
Small unilamellar vesicles have a very low captured volume (approximately 0.5 ml/g) and also a very low encapsulated mass for hydrophilic materials (0.5-1%). However, since the lipid bilayer constitutes 50-87% of the total volume, SUV's are excellent at transporting small quantities of lipophilic material. SUV's primary advantage is in transport of very small quantities of hydrophilic material to tissues where the MLV's or LUV's cannot reach.
Other problems associated with multilamellar lipid vesicles (including the small unilamellar vesiscles which are normally manufactured by sonication of the multilamellar vesicles) are the time of manufacture and expense. Using standard methods, the current processes are both slow and relatively inefficient in terms of material, leading to large expense problems because of the high cost of starter materials. For example, the presently used methods take 2-20 hours to manufacture multilamellar lipid vesicles, and the sonication required to break the multilamellar lipid structures into SUV's takes additional time. This slow processing is unwieldy and expensive for any large scale use of lipid vesicles.
LUV's were developed because of the problems in encapsulating large volumes and obtaining high encapsulated mass for hydrophilic materials. LUV's have large captured volumes (approximately 6-35 ml/g lipid) and high encapsulated mass for hydrophilic materials (70-80%), including macromolecules, but the large relative aqueous volume makes them not as efficient in encapsulating hydrophilic or lipophilic materials as MLV's. In fact LUV's have several problems, even for hydrophilic encapsulation. Since there is only a single lipid bilayer surrounding a large aqueous center, LUV's tend to be less stable then the other lipid vesicles and more easily subject to chemical degradation. Further, the low lipid/aqueous volume ratio makes it difficult to use LUV's for transport of, or targeting with, any lipophilic materials.
Accordingly, an object of the invention is to provide improved lipid vesicles using different materials than those previously known.
Another object of the invention is to provide a method for making lipid vesicles from materials which could not otherwise be used to form the vesicles.
A further object of the invention is to provide inexpensive lipid vesicles which have high uptake of liquid and hydrophilic materials and do not have problems of stability or excessive cost.
A still further object of the invention is to provide inexpensive lipid vesicles which could be used to carry a variety of hydrophilic or lipophilic materials.
These and other objects and features of the invention will be apparent from the following summary of the invention and the description.